Activation of HCV-specific T cells

ABSTRACT

The invention provides a method of activating hepatitis C virus (HCV)-specific T cells, including CD4 +  and CD8 +  T cells. HCV-specific T cells are activated using fusion proteins comprising HCV NS3, NS4, NS5a, and NS5b polypeptides, polynucleotides encoding such fusion proteins, or polypeptide or polynucleotide compositions containing the individual components of these fusions. The method can be used in model systems to develop HCV-specific immunogenic compositions, as well as to immunize a mammal against HCV.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to provisional patent application serial No.60/161,713, filed Oct. 27, 1999, from which priority is claimed under 35USC §119(e)(1) and which is incorporated herein by reference in itsentirety.

TECHNICAL AREA OF THE INVENTION

The invention relates to the activation of hepatitis Cvirus(HCV)-specific T cells. More particularly, the invention relates tothe use of multiple HCV polypeptides, either alone or as fusions, tostimulate cell-mediated immune responses, such as to activateHCV-specific T cells.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) infection is an important health problem withapproximately 1% of the world's population infected with the virus. Over75% of acutely infected individuals eventually progress to a chroniccarrier state that can result in cirrhosis, liver failure, andhepatocellular carcinoma. See Alter et al. (1992) N. Engl. J. Med.327:1899-1905; Resnick and Koff. (1993) Arch. Intem. Med. 153:1672-1677;Seeff (1995) Gastrointest. Dis. 6:20-27; Tong et al. (1995) N. Engl. J.Med. 332:1463-1466.

Despite extensive advances in the development of pharmaceuticals againstcertain viruses like HIV, control of acute and chronic HCV infection hashad limited success (Hooffiagle and di Bisceglie (1997) N. Engl. J. Med.336:347-356). In particular, generation of a strong cytotoxic Tlymphocyte (CTL) response is thought to be important for the control anderadication of HCV infections. Thus, there is a need in the art foreffective methods of inducing strong CTL responses against HCV.

SUMMARY OF THE INVENTION

It is an object of the invention to provide reagents and methods foractivating T cells which recognize epitopes of HCV polypeptides. Thisand other objects of the invention are provided by one or more of theembodiments described below.

The invention provides HCV proteins useful for activating HCV-specific Tcells. One embodiment provides a fusion protein that consistsessentially of an NS3, an NS4, and an NS5a polypeptide.

Another embodiment provides a fusion protein that consists essentiallyof an NS3, an NS4, an NS5a, and NS5b polypeptide of an HCV.

Still another embodiment of the invention provides a fusion proteincomprising an NS3, an NS4, an NS5a and optionally an NS5b polypeptide ofan HCV. One of the HCV polypeptides is derived from a different strainof HCV than the other polypeptides.

The invention also provides compositions comprising any of these fusionproteins and a pharmaceutically acceptable carrier.

Another embodiment provides a composition consisting essentially of anNS3, an NS4, and an NS5a polypeptide, or a composition consistingessentially of polynucleotides encoding the individual proteins.

Another embodiment provides a composition that consists essentially ofan NS3, an NS4, an NS5a, and NS5b polypeptide of an HCV, or acomposition consisting essentially of polynucleotides encoding theindividual proteins.

Still another embodiment of the invention provides a compositionconsisting essentially of an NS3, an NS4, an NS5a and optionally an NS5bpolypeptide of an HCV, or a composition consisting essentially ofpolynucleotides encoding the individual proteins. One of the HCVpolypeptides or polynucleotides is derived from a different strain ofHCV than the others.

Even another embodiment of the invention provides an isolated andpurified polynucleotide which encodes a fusion protein consistingessentially of an NS3, an NS4, and an NS5a polypeptide of an HCV or afusion protein consisting essentially of an NS3, an NS4, an NS5a, and anNS5b polypeptide of an HCV.

Yet another embodiment of the invention provides a compositioncomprising an isolated and purified polynucleotide which encodes afusion protein consisting essentially of either an NS3, an NS4, and anNS5a polypeptide of an HCV or consisting essentially of an NS3, an NS4,an NS5a, and an NS5b polypeptide of an HCV. The composition alsocomprises a pharmaceutically acceptable carrier.

Another embodiment of the invention provides isolated and purifiedpolynucleotide which encodes a fusion protein comprising an NS3, an NS4,and an NS5a polypeptide of an HCV in which one of the NS3, NS4, and NS5apolypeptides is derived from a different strain of HCV than the othertwo polypeptides. The invention also provides a composition comprisingthis polynucleotide and a pharmaceutically acceptable carrier.

Yet another embodiment of the invention provides an isolated andpurified polynucleotide which encodes a fusion protein comprising anNS3, an NS4, an NS5a, and an NS5b polypeptide of an HCV. One of thepolypeptides is derived from a different strain of HCV than the otherpolypeptides. The invention also provides a composition comprising thispolynucleotide and a pharmaceutically acceptable carrier.

Even another embodiment of the invention provides a method of activatingT cells which recognize an epitope of an HCV polypeptide. T cells arecontacted with a fusion protein comprising an NS3, an NS4, and an NS5apolypeptide of an HCV. A population of activated T cells recognizes anepitope of the NS3, NS4, or NS5a polypeptide. Alternatively, T cells arecontacted with a fusion protein comprising an HCV NS3, NS4, NS5a, NS5bpolypeptide of an HCV. A population of activated T cells recognizes anepitope of the NS3, NS4, NS5a, or NS5b polypeptide.

The invention thus provides methods and reagents for activating T cellswhich recognize epitopes of HCV polypeptides. These methods and reagentsare particularly advantageous for identifying epitopes of HCVpolypeptides associated with a strong CTL response and for immunizingmammals, including humans, against HCV.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a side-by-side comparison of IFN-γ expression generated inanimals in response to delivery of alphavirus constructs encodingNS3NS4NS5a.

FIG. 2 shows IFN-γ expression generated in animals in response todelivery of plasmid DNA encoding NS3NS4NS5a (“naked”), PLG-linked DNAencoding NS3NS4NS5a (“PLG”), separate DNA plasmids encoding NS5a, NS34a,and NS4ab (“naked”), and PLG-linked DNA encoding NS5a, NS34a, and NS4ab(“PLG”).

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, recombinantDNA techniques and immunology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al., Molecular Cloning: A Laboratory Manual (2nd Edition); Methods InEnzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); DNACloning, Vols. I and II (D. N. Glover ed.); Oligonucleotide Synthesis(M. J. Gait ed.); Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins eds.); Animal Cell Culture (R. K. Freshney ed.); Perbal, B., APractical Guide to Molecular Cloning.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “an antigen” includes a mixture of two or more antigens,and the like.

I. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also include postexpressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation and the like. Furthermore, for purposes ofthe present invention, a “polypeptide” refers to a protein whichincludes modifications, such as deletions, additions and substitutions(generally conservative in nature), to the native sequence, so long asthe protein maintains the desired activity. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts which produce the proteins or errorsdue to PCR amplification.

An HCV polypeptide is a polypeptide, as defined above, derived from theHCV polyprotein. The polypeptide need not be physically derived fromHCV, but may be synthetically or recombinantly produced. Moreover, thepolypeptide may be derived from any of the various HCV strains, such asfrom strains 1, 2, 3 or 4 of HCV. A number of conserved and variableregions are known between these strains and, in general, the amino acidsequences of epitopes derived from these regions will have a high degreeof sequence homology, e.g., amino acid sequence homology of more than30%, preferably more than 40%, when the two sequences are aligned. Thus,for example, the term “NS4” polypeptide refers to native NS4 from any ofthe various HCV strains, as well as NS4 analogs, muteins and immunogenicfragments, as defined further below.

The terms “analog” and “mutein” refer to biologically active derivativesof the reference molecule, or fragments of such derivatives, that retaindesired activity, such as the ability to stimulate a cell-mediatedimmune response, as defined below. In general, the term “analog” refersto compounds having a native polypeptide sequence and structure with oneor more amino acid additions, substitutions (generally conservative innature) and/or deletions, relative to the native molecule, so long asthe modifications do not destroy immunogenic activity. The term “mutein”refers to peptides having one or more peptide mimics (“peptoids”), suchas those described in International Publication No. WO 91/04282.Preferably, the analog or mutein has at least the same immunoactivity asthe native molecule. Methods for making polypeptide analogs and muteinsare known in the art and are described further below.

Particularly preferred analogs include substitutions that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, an aspartatewith a glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid,will not have a major effect on the biological activity. For example,the polypeptide of interest may include up to about 5-10 conservative ornon-conservative amino acid substitutions, or even up to about 15-25conservative or non-conservative amino acid substitutions, or anyinteger between 5-25, so long as the desired function of the moleculeremains intact. One of skill in the art may readily determine regions ofthe molecule of interest that can tolerate change by reference toHopp/Woods and Kyte-Doolittle plots, well known in the art.

By “fragment” is intended a polypeptide consisting of only a part of theintact full-length polypeptide sequence and structure. The fragment caninclude a C-terminal deletion and/or an N-terminal deletion of thenative polypeptide. An “immunogenic fragment” of a particular HCVprotein will generally include at least about 5-10 contiguous amino acidresidues of the full-length molecule, preferably at least about 15-25contiguous amino acid residues of the full-length molecule, and mostpreferably at least about 20-50 or more contiguous amino acid residuesof the full-length molecule, that define an epitope, or any integerbetween 5 amino acids and the full-length sequence, provided that thefragment in question retains immunogenic activity, as measured by theassays described herein. For a description of various HCV epitopes, see,e.g., Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015;Chien et al., J. Gastroent. Hepatol. (1993) 8:S33-39; Chien et al.,International Publication No. WO 93/00365; Chien, D. Y., InternationalPublication No. WO 94/01778; commonly owned, allowed U.S. patentapplication Ser. Nos. 08/403,590 and 08/444,818.

The term “epitope” as used herein refers to a sequence of at least about3 to 5, preferably about 5 to 10 or 15, and not more than about 1,000amino acids (or any integer therebetween), which define a sequence thatby itself or as part of a larger sequence, binds to an antibodygenerated in response to such sequence. There is no critical upper limitto the length of the fragment, which may comprise nearly the full-lengthof the protein sequence, or even a fusion protein comprising two or moreepitopes from the HCV polyprotein. An epitope for use in the subjectinvention is not limited to a polypeptide having the exact sequence ofthe portion of the parent protein from which it is derived. Indeed,viral genomes are in a state of constant flux and contain severalvariable domains which exhibit relatively high degrees of variabilitybetween isolates. Thus the term “epitope” encompasses sequencesidentical to the native sequence, as well as modifications to the nativesequence, such as deletions, additions and substitutions (generallyconservative in nature).

Regions of a given polypeptide that include an epitope can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. Forexample, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, allincorporated herein by reference in their entireties. Similarly,conformational epitopes are readily identified by determining spatialconformation of amino acids such as by, e.g., x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols, supra. Antigenic regions of proteins can also be identifiedusing standard antigenicity and hydropathy plots, such as thosecalculated using, e.g., the Omiga version 1.0 software program availablefrom the Oxford Molecular Group. This computer program employs theHopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981)78:3824-3828 for determining antigenicity profiles, and theKyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132for hydropathy plots.

As used herein, the term “conformational epitope” refers to a portion ofa full-length protein, or an analog or mutein thereof, having structuralfeatures native to the amino acid sequence encoding the epitope withinthe full-length natural protein. Native structural features include, butare not limited to, glycosylation and three dimensional structure.Preferably, a conformational epitope is produced recombinantly and isexpressed in a cell from which it is extractable under conditions whichpreserve its desired structural features, e.g. without denaturation ofthe epitope. Such cells include bacteria, yeast, insect, and mammaliancells. Expression and isolation of recombinant conformational epitopesfrom the HCV polyprotein are described in e.g., InternationalPublication Nos. WO 96/04301, WO 94/01778, WO 95/33053, WO 92/08734,which applications are herein incorporated by reference in theirentirety.

An “immunological response” to an HCV antigen (including bothpolypeptide and polynucleotides encoding polypeptides that are expressedin vivo) or composition is the development in a subject of a humoraland/or a cellular immune response to molecules present in thecomposition of interest. For purposes of the present invention, a“humoral immune response” refers to an immune response mediated byantibody molecules, while a “cellular immune response” is one mediatedby T-lymphocytes and/or other white blood cells. One important aspect ofcellular immunity involves an antigen-specific response by cytolyticT-cells (“CTLs”). CTLs have specificity for peptide antigens that arepresented in association with proteins encoded by the majorhistocompatibility complex (MHC) and expressed on the surfaces of cells.CTLs help induce and promote the intracellular destruction ofintracellular microbes, or the lysis of cells infected with suchmicrobes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376; and the examples below.

Thus, an immunological response as used herein may be one whichstimulates the production of CTLs, and/or the production or activationof helper T-cells. The antigen of interest may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by B-cells; and/or the activation of suppressor T-cellsand/or γδ T-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest. These responses mayserve to neutralize infectivity, and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection oralleviation of symptoms to an immunized host. Such responses can bedetermined using standard immunoassays and neutralization assays, wellknown in the art.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invitro or in vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxy) terminus. A transcription termination sequence may belocated 3′ to the coding sequence.

A “nucleic acid” molecule or “polynucleotide” can include both double-and single-stranded sequences and refers to, but is not limited to, cDNAfrom viral, procaryotic or eucaryotic mRNA, genomic DNA sequences fromviral (e.g. DNA viruses and retroviruses) or procaryotic DNA, andespecially synthetic DNA sequences. The term also captures sequencesthat include any of the known base analogs of DNA and RNA.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their desiredfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper transcription factors, etc., are present. The promoter need notbe contiguous with the coding sequence, so long as it functions todirect the expression thereof. Thus, for example, interveninguntranslated yet transcribed sequences can be present between thepromoter sequence and the coding sequence, as can transcribed introns,and the promoter sequence can still be considered “operably linked” tothe coding sequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, viral, semisynthetic, or syntheticorigin which, by virtue of its origin or manipulation is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide. In general, the gene of interest is cloned and thenexpressed in transformed organisms, as described further below. The hostorganism expresses the foreign gene to produce the protein underexpression conditions.

A “control element” refers to a polynucleotide sequence which aids inthe expression of a coding sequence to which it is linked. The termincludes promoters, transcription termination sequences, upstreamregulatory domains, polyadenylation signals, untranslated regions,including 5′-UTRs and 3′-UTRs and when appropriate, leader sequences andenhancers, which collectively provide for the transcription andtranslation of a coding sequence in a host cell.

A “promoter” as used herein is a DNA regulatory region capable ofbinding RNA polymerase in a host cell and initiating transcription of adownstream (3′ direction) coding sequence operably linked thereto. Forpurposes of the present invention, a promoter sequence includes theminimum number of bases or elements necessary to initiate transcriptionof a gene of interest at levels detectable above background. Within thepromoter sequence is a transcription initiation site, as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. Eucaryotic promoters will often, but not always, contain“TATA” boxes and “CAT” boxes.

A control sequence “directs the transcription” of a coding sequence in acell when RNA polymerase will bind the promoter sequence and transcribethe coding sequence into mRNA, which is then translated into thepolypeptide encoded by the coding sequence.

“Expression cassette” or “expression construct” refers to an assemblywhich is capable of directing the expression of the sequence(s) orgene(s) of interest. The expression cassette includes control elements,as described above, such as a promoter which is operably linked to (soas to direct transcription of) the sequence(s) or gene(s) of interest,and often includes a polyadenylation sequence as well. Within certainembodiments of the invention, the expression cassette described hereinmay be contained within a plasmid construct. In addition to thecomponents of the expression cassette, the plasmid construct may alsoinclude, one or more selectable markers, a signal which allows theplasmid construct to exist as single-stranded DNA (e.g., a M13 origin ofreplication), at least one multiple cloning site, and a “mammalian”origin of replication (e.g., a SV40 or adenovirus origin ofreplication).

“Transformation,” as used herein, refers to the insertion of anexogenous polynucleotide into a host cell, irrespective of the methodused for insertion: for example, transformation by direct uptake,transfection, infection, and the like. For particular methods oftransfection, see further below. The exogenous polynucleotide may bemaintained as a nonintegrated vector, for example, an episome, oralternatively, may be integrated into the host genome.

A “host cell” is a cell which has been transformed, or is capable oftransformation, by an exogenous DNA sequence.

By “isolated” is meant, when referring to a polypeptide, that theindicated molecule is separate and discrete from the whole organism withwhich the molecule is found in nature or is present in the substantialabsence of other biological macro-molecules of the same type. The term“isolated” with respect to a polynucleotide is a nucleic acid moleculedevoid, in whole or part, of sequences normally associated with it innature; or a sequence, as it exists in nature, but having heterologoussequences in association therewith; or a molecule disassociated from thechromosome.

The term “purified” as used herein preferably means at least 75% byweight, more preferably at least 85% by weight, more preferably still atleast 95% by weight, and most preferably at least 98% by weight, ofbiological macromolecules of the same type are present.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when the sequences exhibit atleast about 50%, preferably at least about 75%, more preferably at leastabout 80%-85%, preferably at least about 90%, and most preferably atleast about 95%-98%, or more, sequence identity over a defined length ofthe molecules. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100. Readily available computerprograms can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5Suppl. 3:353-358, National biomedical Research Foundation, Washington,D.C., which adapts the local homology algorithm of Smith and WatermanAdvances in Appl. Math. 2:482-489, 1981 for peptide analysis. Programsfor determining nucleotide sequence identity are available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAPprograms, which also rely on the Smith and Waterman algorithm. Theseprograms are readily utilized with the default parameters recommended bythe manufacturer and described in the Wisconsin Sequence AnalysisPackage referred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Another method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

By “nucleic acid immunization” is meant the introduction of a nucleicacid molecule encoding one or more selected antigens into a host cell,for the in vivo expression of the antigen or antigens. The nucleic acidmolecule can be introduced directly into the recipient subject, such asby injection, inhalation, oral, intranasal and mucosal administration,or the like, or can be introduced ex vivo, into cells which have beenremoved from the host. In the latter case, the transformed cells arereintroduced into the subject where an immune response can be mountedagainst the antigen encoded by the nucleic acid molecule.

As used herein, “treatment” refers to any of (i) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

By “vertebrate subject” is meant any member of the subphylum cordata,including, without limitation, humans and other primates, includingnon-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The term does not denote a particularage. Thus, both adult and newborn individuals are intended to becovered. The invention described herein is intended for use in any ofthe above vertebrate species, since the immune systems of all of thesevertebrates operate similarly.

II. Modes of Carrying Out the Invention

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of compositions and methods similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

It is a discovery of the present invention that fusion proteins,combinations of the individual components of these fusions, andpolynucleotides encoding the same, comprising an NS3, an NS4, and anNS5a polypeptide or an NS3, an NS4, an NS5a, and an NS5b polypeptide ofan HCV virus can be used to activate HCV-specific T cells, i.e., T cellswhich recognize epitopes of these polypeptides. Activation ofHCV-specific T cells by such fusion polypeptides and proteins, orcombinations of the individual polypeptides that make up the fusions,provide both in vitro and in vivo model systems for the development ofHCV vaccines, particularly for identifying HCV polypeptide epitopesassociated with a response. The fusion proteins, or combinations of theindividual proteins, can also be used to generate an immune responseagainst HCV in a mammal, particularly a CTL response for eithertherapeutic or prophylactic purposes.

NS3NS4NS5a and NS3NS4NS5aNS5b Fusion Proteins

The genomes of HCV strains contain a single open reading frame ofapproximately 9,000 to 12,000 nucleotides, which is transcribed into apolyprotein. An HCV polyprotein is cleaved to produce at least tendistinct products, in the order ofNH₂-Core-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Fusion proteins ofthe invention (NS3NS4NS5a fusion proteins, also termed “NS345a” herein)comprise HCV NS3, NS4 (NS4a and NS4b), and NS5a polypeptides or compriseHCV NS3, NS4 (NS4a and NS4b), NS5a, and NS5b polypeptides(NS3NS4NS5aNS5b fusion proteins, also termed “NS345ab” herein). Otherfusions described in the examples include fusions of HCV NS3 and NS4(NS4a and NS4b, also termed “NS34” and “NS34ab”); as well as fusions ofHCV NS3 and NS4a (also termed “NS34a”).

The HCV NS3 protein functions as a protease and a helicase and occurs atapproximately amino acid 1027 to amino acid 1657 of the polyprotein(numbered relative to HCV-1). See Choo et al. (1991) Proc. Natl. Acad.Sci. USA 88:2451-2455. HCV NS4 occurs at approximately amino acid 1658to amino acid 1972, NS5a occurs at approximately amino acid 1973 toamino acid 2420, and HCV NS5b occurs at approximately amino acid 2421 toamino acid 3011 of the polyprotein (numbered relative to HCV-1) (Choo etal., 1991).

The NS3, NS4, NS5a, and NS5b polypeptides present in the various fusionsdescribed above can either be full-length polypeptides or portions ofNS3, NS4 (NS4a and NS4b), NS5a, and NS5b polypeptides. The portions ofNS3, NS4, NS5a, and NS5b polypeptides making up the fusion proteincomprise at least one epitope, which is recognized by a T cell receptoron an activated T cell, such as 2152-HEYPVGSQL-2160 (SEQ ID NO:1) and2224-AELIEANLLWRQEMG-2238 (SEQ ID NO:2). Epitopes of NS3, NS4 (NS4a andNS4b), NS5a, NS5b, NS3NS4NS5a, and NS3NS4NS5aNS5b can be identified byseveral methods. For example, NS3, NS4, NS5a, NS5b polypeptides orfusion proteins comprising any combination of the above, can beisolated, for example, by immunoaffinity purification using a monoclonalantibody for the polypeptide or protein. The isolated protein sequencecan then be screened by preparing a series of short peptides byproteolytic cleavage of the purified protein, which together span theentire protein sequence. By starting with, for example, 100-merpolypeptides, each polypeptide can be tested for the presence ofepitopes recognized by a T cell receptor on an HCV-activated T cell,progressively smaller and overlapping fragments can then be tested froman identified 100-mer to map the epitope of interest.

Epitopes recognized by a T cell receptor on an HCV-activated T cell canbe identified by, for example, ⁵¹Cr release assay (see Example 2) or bylymphoproliferation assay (see Example 4). In a ⁵¹Cr release assay,target cells can be constructed that display the epitope of interest bycloning a polynucleotide encoding the epitope into an expression vectorand transforming the expression vector into the target cells.HCV-specific CD8⁺ T cells will lyse target cells displaying an NS3, NS4,NS5a, NS5b, NS3NS4NS5a, or NS3NS4NS5aNS5b epitope and will not lysecells that do not display such an epitope. In an lymphoproliferationassay, HCV-activated CD4⁺ T cells will proliferate when cultured with anNS3, NS4, NS5a, NS5b, NS3NS4NS5a, or NS3NS4NS5aNS5b epitopic peptide,but not in the absence of an HCV epitopic peptide.

NS3, NS4, NS5a, and NS5b polypeptides can occur in any order in thefusion protein. If desired, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore of one or more of the polypeptides may occur in the fusion protein.Multiple viral strains of HCV occur, and NS3, NS4, NS5a, and NS5bpolypeptides of any of these strains can be used in a fusion protein.

Nucleic acid and amino acid sequences of a number of HCV strains andisolates, including nucleic acid and amino acid sequences of NS3, NS4,NS5a, NS5b genes and polypeptides have been determined. For example,isolate HCV J1.1 is described in Kubo et al. (1989) Japan. Nucl. AcidsRes. 17:10367-10372; Takeuchi et al. (1990) Gene 91:287-291; Takeuchi etal. (1990) J. Gen. Virol. 71:3027-3033; and Takeuchi et al. (1990) Nucl.Acids Res. 18:4626. The complete coding sequences of two independentisolates, HCV-J and BK, are described by Kato et al., (1990) Proc. Natl.Acad. Sci. USA 87:9524-9528 and Takamizawa et al., (1991) J. Virol.65:1105-1113 respectively.

Publications that describe HCV-1 isolates include Choo et al. (1990)Brit. Med. Bull. 46:423-441; Choo et al. (1991) Proc. Natl. Acad. Sci.USA 88:2451-2455 and Han et al. (1991) Proc. Natl. Acad. Sci. USA88:1711-1715. HCV isolates HC-J1 and HC-J4 are described in Okamoto etal. (1991) Japan J. Exp. Med. 60:167-177. HCV isolates HCT 18˜, HCT 23,Th, HCT 27, EC1 and EC10 are described in Weiner et al. (1991) Virol.180:842-848. HCV isolates Pt-1, HCV-K1 and HCV-K2 are described inEnomoto et al. (1990) Biochem. Biophys. Res. Commun. 170:1021-1025. HCVisolates A, C, D & E are described in Tsukiyama-Kohara et al. (1991)Virus Genes 5:243-254.

Each of the NS3, NS4, NS5a, and NS5b components of a fusion protein canbe obtained from the same HCV strain or isolate or from different HCVstrains or isolates. Fusion proteins comprising HCV polypeptides from,for example, the NS3 polypeptide can be derived from a first strain ofHCV, and the NS4, and NS5a polypeptides can be derived from a secondstrain of HCV. Alternatively, the NS4 polypeptide can be derived from afirst strain of HCV, and the NS3 and NS5a polypeptides can be derivedfrom a second strain of HCV. Optionally, the NS5a polypeptide can bederived from a first strain of HCV, and the NS3 and NS4 polypeptides canbe derived from a second strain of HCV, NS3, NS4 and NS5a polypeptidesthat are each be derived from different HCV strains can also be used inan NS3NS4NS5a fusion protein. Similarly, in a fusion protein comprisingNS5b, at least one of the NS3, NS4, NS5a, and NS5b polypeptides can bederived from a different HCV strain than the other polypeptides.Optionally, NS3, NS4, NS5a, and NS5b polypeptides that are each derivedfrom different HCV strains can also be used in an NS3NS4NS5aNS5b fusionprotein.

In addition to NS3, NS4a, NS4b, NS5a and NS5b, the fusion proteins cancontain other polypeptides derived from the HCV polyprotein. Forexample, it may be desirable to include polypeptides derived from thecore region of the HCV polyprotein. This region occurs at amino acidpositions 1-191 of the HCV polyprotein, numbered relative to HCV-1.Either the full-length protein or epitopes of the full-length proteinmay be used in the subject fusions, such as those epitopes found betweenamino acids 10-53, amino acids 10-45, amino acids 67-88, amino acids120-130, or any of the core epitopes identified in, e.g., Houghton etal., U.S. Pat. No. 5,350,671; Chien et al., Proc. Natl. Acad. Sci. USA(1992) 89:10011-10015; Chien et al., J. Gastroent. Hepatol. (1993)8:S33-39; Chien et al., International Publication No. WO 93/00365;Chien, D. Y., International Publication No. WO 94/01778; and commonlyowned, allowed U.S. patent application Ser. Nos. 08/403,590 and08/444,818, the disclosures of which are incorporated herein byreference in their entireties.

Preferably, the above-described fusion proteins, as well as theindividual components of these proteins, are produced recombinantly. Apolynucleotide encoding these proteins can be introduced into anexpression vector which can be expressed in a suitable expressionsystem. A variety of bacterial, yeast, mammalian and insect expressionsystems are available in the art and any such expression system can beused. Optionally, a polynucleotide encoding these proteins can betranslated in a cell-free translation system. Such methods are wellknown in the art. The proteins also can be constructed by solid phaseprotein synthesis.

If desired, the fusion proteins, or the individual components of theseproteins, also can contain other amino acid sequences, such as aminoacid linkers or signal sequences, as well as ligands useful in proteinpurification, such as glutathione-S-transferase and staphylococcalprotein A.

NS3NS4NS5a and NS3NS4NS5aNS5b Polynucleotides

Polynucleotides contain less than an entire HCV genome and can be RNA orsingle- or double-stranded DNA. Preferably, the polynucleotides areisolated free of other components, such as proteins and lipids.NS3NS4NS5a polynucleotides encode the NS3NS4NS5a fusion proteinsdescribed above, and thus comprise coding sequences for NS3, NS4, andNS5a polypeptides. NS3NS4NS5aNS5b polynucleotides encode theNS3NS4NS5aNS5b fusion proteins described above, and thus comprise codingsequences for NS3, NS4, NS5a, and NS5b polypeptides. Similarly,polynucleotides encoding other fusions, such as NS3NS4 and NS3NS4a willcomprise sequences for the individual HCV polypeptides. Polynucleotidesof the invention can also comprise other nucleotide sequences, such assequences coding for linkers, signal sequences, or ligands useful inprotein purification such as glutathione-S-transferase andstaphylococcal protein A.

Polynucleotides encoding NS3, NS4, NS5a and/or NS5b can be isolated froma genomic library derived from nucleic acid sequences present in, forexample, the plasma, serum, or liver homogenate of an HCV infectedindividual or can be synthesized in the laboratory, for example, usingan automatic synthesizer. An amplification method such as PCR can beused to amplify polynucleotides from either HCV genomic DNA or cDNAencoding NS3, NS4, NS5a, or NS5b.

Polynucleotides encoding NS3, NS4, NS5a, or NS5b polypeptides cancomprise coding sequences for these polypeptides which occur naturallyor can be artificial sequences which do not occur in nature. Thesepolynucleotides can be ligated to form a coding sequence for the fusionproteins using standard molecular biology techniques. If desired,polynucleotides can be cloned into an expression vector and transformedinto, for example, bacterial, yeast, insect, or mammalian cells so thatthe fusion proteins of the invention can be expressed in and isolatedfrom a cell culture.

The expression constructs of the present invention, including thedesired fusion, or individual expression constructs comprising theindividual components of these fusions, may be used for nucleic acidimmunization, to activate HCV-specific T cells, using standard genedelivery protocols. Methods for gene delivery are known in the art. See,e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. Genes can be delivered eitherdirectly to the vertebrate subject or, alternatively, delivered ex vivo,to cells derived from the subject and the cells reimplanted in thesubject. For example, the constructs can be delivered as plasmid DNA,e.g., contained within a plasmid, such as pBR322, pUC, or ColE1Additionally, the expression constructs can be packaged in liposomesprior to delivery to the cells. Lipid encapsulation is generallyaccomplished using liposomes which are able to stably bind or entrap andretain nucleic acid. The ratio of condensed DNA to lipid preparation canvary but will generally be around 1:1 (mg DNA:micromoles lipid), or moreof lipid. For a review of the use of liposomes as carriers for deliveryof nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991)1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol.101, pp. 512-527.

Liposomal preparations for use with the present invention includecationic (positively charged), anionic (negatively charged) and neutralpreparations, with cationic liposomes particularly preferred. Cationicliposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987)84:7413-7416). Other commercially available lipids include transfectace(DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can beprepared from readily available materials using techniques well known inthe art. See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978)75:4194-4198; PCT Publication No. WO 90/11092 for a description of thesynthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)liposomes. The various liposome-nucleic acid complexes are preparedusing methods known in the art. See, e.g., Straubinger et al., inMETHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al., Proc.Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et al.,Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell (1979)17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629; Ostroet al., Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al.,Proc. Natl. Acad. Sci. USA (1979) 76:3348); Enoch and Strittmatter,Proc. Natl. Acad. Sci. USA (1979) 76: 145); Fraley et al., J. Biol.Chem. (1980) 255:10431; Szoka and Papahadjopoulos, Proc. Natl. Acad.Sci. USA (1978) 75:145; and Schaefer-Ridder et al., Science (1982)215:166.

The DNA can also be delivered in cochleate lipid compositions similar tothose described by Papahadjopoulos et al., Biochem. Biophys. Acta.(1975) 394:483-491. See, also, U.S. Pat. Nos. 4,663,161 and 4,871,488.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems, such as murine sarcoma virus, mousemammary tumor virus, Moloney murine leukemia virus, and Rous sarcomavirus. A selected gene can be inserted into a vector and packaged inretroviral particles using techniques known in the art. The recombinantvirus can then be isolated and delivered to cells of the subject eitherin vivo or ex vivo. A number of retroviral systems have been described(U.S. Pat. No. 5,219,740; Miller and Rosman, BioTechniques (1989)7:980-990; Miller, A. D., Human Gene Therapy (1990) 1:5-14; Scarpa etal., Virology (1991) 180:849-852; Burns et al., Proc. Natl. Acad. Sci.USA (1993) 90:8033-8037; and Boris-Lawrie and Temin, Cur. Opin. Genet.Develop. (1993) 3:102-109. Briefly, retroviral gene delivery vehicles ofthe present invention may be readily constructed from a wide variety ofretroviruses, including for example, B, C, and D type retroviruses aswell as spumaviruses and lentiviruses such as FIV, HIV, HIV-1, HIV-2 andSIV (see RNA Tumor Viruses, Second Edition, Cold Spring HarborLaboratory, 1985). Such retroviruses may be readily obtained fromdepositories or collections such as the American Type Culture Collection(“ATCC”; 10801 University Blvd., Manassas, Va. 20110-2209), or isolatedfrom known sources using commonly available techniques.

A number of adenovirus vectors have also been described, such asadenovirus Type 2 and Type 5 vectors. Unlike retroviruses whichintegrate into the host genome, adenoviruses persist extrachromosomallythus minimizing the risks associated with insertional mutagenesis(Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J.Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994)5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., GeneTherapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988) 6:616-629;and Rich et al., Human Gene Therapy (1993) 4:461-476).

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 andWagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can alsobe used for gene delivery.

Members of the Alphavirus genus, such as but not limited to vectorsderived from the Sindbis and Semliki Forest viruses, VEE, will also finduse as viral vectors for delivering the gene of interest. For adescription of Sindbis-virus derived vectors useful for the practice ofthe instant methods, see, Dubensky et al., J. Virol. (1996) 70:508-519;and International Publication Nos. WO 95/07995 and WO 96/17072.

Other vectors can be used, including but not limited to simian virus 40,cytomegalovirus. Bacterial vectors, such as Salmonella ssp. Yersiniaenterocolitica, Shigella spp., Vibrio cholerae, Mycobacterium strainBCG, and Listeria monocytogenes can be used. Minichromosomes such as MCand MC1, bacteriophages, cosmids (plasmids into which phage lambda cossites have been inserted) and replicons (genetic elements that arecapable of replication under their own control in a cell) can also beused.

The expression constructs may also be encapsulated, adsorbed to, orassociated with, particulate carriers. Such carriers present multiplecopies of a selected molecule to the immune system and promote trappingand retention of molecules in local lymph nodes. The particles can bephagocytosed by macrophages and can enhance antigen presentation throughcytokine release. Examples of particulate carriers include those derivedfrom polymethyl methacrylate polymers, as well as microparticles derivedfrom poly(lactides) and poly(lactide-co-glycolides), known as PLG. See,e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; and McGee et al.,J. Microencap. (1996).

A wide variety of other methods can be used to deliver the expressionconstructs to cells. Such methods include DEAE dextran-mediatedtransfection, calcium phosphate precipitation, polylysine- orpolyornithine-mediated transfection, or precipitation using otherinsoluble inorganic salts, such as strontium phosphate, aluminumsilicates including bentonite and kaolin, chromic oxide, magnesiumsilicate, talc, and the like. Other useful methods of transfectioninclude electroporation, sonoporation, protoplast fusion, liposomes,peptoid delivery, or microinjection. See, e.g., Sambrook et al., supra,for a discussion of techniques for transforming cells of interest; andFelgner, P. L., Advanced Drug Delivery Reviews (1990) 5:163-187, for areview of delivery systems useful for gene transfer. One particularlyeffective method of delivering DNA using electroporation is described inInternational Publication No. WO/0045823.

Additionally, biolistic delivery systems employing particulate carrierssuch as gold and tungsten, are especially useful for delivering theexpression constructs of the present invention. The particles are coatedwith the construct to be delivered and accelerated to high velocity,generally under a reduced atmosphere, using a gun powder discharge froma “gene gun.” For a description of such techniques, and apparatusesuseful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006;5,100,792; 5,179,022; 5,371,015; and 5,478,744.

Compositions Comprising Fusion Proteins or Polynucleotides

The invention also provides compositions comprising the fusion proteinsor polynucleotides, as well as compositions including the individualcomponents of these fusion proteins or polynucleotides. Compositions ofthe invention preferably comprise a pharmaceutically acceptable carrier.The carrier should not itself induce the production of antibodiesharmful to the host. Pharmaceutically acceptable carriers are well knownto those in the art. Such carriers include, but are not limited to,large, slowly metabolized, macromolecules, such as proteins,polysaccharides such as latex functionalized sepharose, agarose,cellulose, cellulose beads and the like, polylactic acids, polyglycolicacids, polymeric amino acids such as polyglutamic acid, polylysine, andthe like, amino acid copolymers, and inactive virus particles.

Pharmaceutically acceptable salts can also be used in compositions ofthe invention, for example, mineral salts such as hydrochlorides,hydrobromides, phosphates, or sulfates, as well as salts of organicacids such as acetates, proprionates, malonates, or benzoates.Especially useful protein substrates are serum albumins, keyhole limpethemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanustoxoid, and other proteins well known to those of skill in the art.Compositions of the invention can also contain liquids or excipients,such as water, saline, glycerol, dextrose, ethanol, or the like, singlyor in combination, as well as substances such as wetting agents,emulsifying agents, or pH buffering agents. Liposomes can also be usedas a carrier for a composition of the invention, such liposomes aredescribed above.

If desired, co-stimulatory molecules which improve immunogenpresentation to lymphocytes, such as B7-1 or B7-2, or cytokines such asGM-CSF, IL-2, and IL-12, can be included in a composition of theinvention. Optionally, adjuvants can also be included in a composition.Adjuvants which can be used include, but are not limited to: (1)aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate,aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with orwithout other specific immunostimulating agents such as muramyl peptides(see below) or bacterial cell wall components), such as for example (a)MF59 (PCT Publ. No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80,and 0.5% Span 85 (optionally containing various amounts of MTP-PE ),formulated into submicron particles using a microfluidizer such as Model110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, andthr-MDP (see below) either microfluidized into a submicron emulsion orvortexed to generate a larger particle size emulsion, and (c) Ribimadjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2%Squalene, 0.2% Tween 80, and one or more bacterial cell wall componentsfrom the group consisting of monophosphorylipid A (MPL), trehalosedimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS(Detox™); (3) saponin adjuvants, such as Stimulon™ (CambridgeBioscience, Worcester, Mass.) may be used or particles generatedtherefrom such as ISCOMs (immunostimulating complexes); (4) CompleteFreund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5)cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; (6)detoxified mutants of a bacterial ADP-ribosylating toxin such as acholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labiletoxin (LT), particularly LT-K63, LT-R72, CT-S109, PT-K9/G129; see, e.g.,WO 93/13302 and WO 92/19265; (7) other substances that act asimmunostimulating agents to enhance the effectiveness of thecomposition; and (8) microparticles with adsorbed macromolecules, asdescribed in copending U.S. patent application Ser. No. 09/285,855(filed Apr. 2, 1999) and international Patent Application Ser. No.PCT/US99/17308 (filed Jul. 29, 1999). Alum and MF59 are preferred.

As mentioned above, muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred tonor-MDP), N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP19835A, referred to as MTP-PE), etc.

Methods of Producing HCV-Specific Antibodies

The HCV fusion proteins, such as NS3NS4SN5a and NS3NS4NS5aNS5b fusionproteins, can be used to produce HCV-specific polyclonal and monoclonalantibodies. HCV-specific polyclonal and monoclonal antibodiesspecifically bind to HCV antigens.

Polyclonal antibodies can be produced by administering the fusionprotein to a mammal, such as a mouse, a rabbit, a goat, or a horse.Serum from the immunized animal is collected and the antibodies arepurified from the plasma by, for example, precipitation with ammoniumsulfate, followed by chromatography, preferably affinity chromatography.Techniques for producing and processing polyclonal antisera are known inthe art.

Monoclonal antibodies directed against HCV-specific epitopes present inthe fusion proteins can also be readily produced. Normal B cells from amammal, such as a mouse, immunized with, e.g., an NS3NS4SN5a or anNS3NS4NS5aNS5b fusion protein can be fused with, for example,HAT-sensitive mouse myeloma cells to produce hybridomas. Hybridomasproducing HCV-specific antibodies can be identified using RIA or ELISAand isolated by cloning in semi-solid agar or by limiting dilution.Clones producing HCV-specific antibodies are isolated by another roundof screening.

Antibodies, either monoclonal and polyclonal, which are directed againstHCV epitopes, are particularly useful for detecting the presence of HCVor HCV antigens in a sample, such as a serum sample from an HCV-infectedhuman. An immunoassay for an HCV antigen may utilize one antibody orseveral antibodies. An immunoassay for an HCV antigen may use, forexample, a monoclonal antibody directed towards an HCV epitope, acombination of monoclonal antibodies directed towards epitopes of oneHCV polypeptide, monoclonal antibodies directed towards epitopes ofdifferent HCV polypeptides, polyclonal antibodies directed towards thesame HCV antigen, polyclonal antibodies directed towards different HCVantigens, or a combination of monoclonal and polyclonal antibodies.Immunoassay protocols may be based, for example, upon competition,direct reaction, or sandwich type assays using, for example, labeledantibody. The labels may be, for example, fluorescent, chemiluminescent,or radioactive.

The polyclonal or monoclonal antibodies may further be used to isolateHCV particles or antigens by immunoaffinity columns. The antibodies canbe affixed to a solid support by, for example, adsorption or by covalentlinkage so that the antibodies retain their immunoselective activity.Optionally, spacer groups may be included so that the antigen bindingsite of the antibody remains accessible. The immobilized antibodies canthen be used to bind HCV particles or antigens from a biological sample,such as blood or plasma. The bound HCV particles or antigens arerecovered from the column matrix by, for example, a change in pH.

HCV-Specific T cells

HCV-specific T cells that are activated by the above-described fusions,including the NS3NS4NS5a fusion protein or NS3NS4NS5aNS5b fusionprotein, expressed in vivo or in vitro, or combinations of theindividual components of the fusions, preferably recognize an epitope ofan HCV polypeptide such as an NS3, NS4, NS5a, NS5b polypeptide,including an epitope of an NS3NS4NS5a fusion protein or anNS3NS4NS5aNS5b fusion protein. HCV-specific T cells can be CD8⁺ or CD4⁺.

HCV-specific CD8⁺ T cells preferably are cytotoxic T lymphocytes (CTL)which can kill HCV-infected cells that display NS3, NS4, NS5a, NS5bepitopes complexed with an MHC class I molecule. HCV-specific CD8⁺ Tcells may also express interferon-γ (IFN-γ). HCV-specific CD8⁺ T cellscan be detected by, for example, ⁵¹Cr release assays (see Example 2).⁵¹Cr release assays measure the ability of HCV-specific CD8⁺ T cells tolyse target cells displaying an NS3, NS4, NS5a, NS5b, NS3NS4NS5a, orNS3NS4NS5aNS5b epitope. HCV-specific CD8⁺ T cells which express IFN-γcan also be detected by immunological methods, preferably byintracellular staining for IFN-γ after in vitro stimulation with an NS3,an NS4, an NS5a, or an NS5b polypeptide (see Example 3).

HCV-specific CD4⁺ cells activated by the above-described fusions, suchas an NS3NS4NS5a or NS3NS4NS5aNS5b fusion protein, expressed in vivo orin vitro, and combinations of the individual components of theseproteins, preferably recognize an epitope of an NS3, NS4, NS5a, or NS5bpolypeptide, including an epitope of an NS3NS4NS5a or NS3NS4NS5aNS5bfusion protein, that is bound to an MHC class II molecule on anHCV-infected cell and proliferate in response to stimulating NS3NS4NS5aor NS3NS4NS5aNS5b peptides.

HCV-specific CD4⁺ T cells can be detected by a lymphoproliferation assay(see Example 4). Lymphoproliferation assays measure the ability ofHCV-specific CD4⁺ T cells to proliferate in response to an NS3, an NS4,an NS5a, or an NS5b epitope.

Methods of Activating HCV-Specific T Cells

NS3NS4NS5a fusion proteins or polynucleotides and NS3NS4NS5aNS5b fusionproteins or polynucleotides, or combinations of the individualcomponents of these proteins and polynucleotides, can be used toactivate HCV-specific T cells either in vitro or in vivo. Activation ofHCV-specific T cells can be used, inter alia, to provide model systemsto optimize CTL responses to HCV and to provide prophylactic ortherapeutic treatment against HCV infection. For in vitro activation,proteins are preferably supplied to T cells via a plasmid or a viralvector, such as an adenovirus vector, as described above.

Polyclonal populations of T cells can be derived from the blood, andpreferably from peripheral lymphoid organs, such as lymph nodes, spleen,or thymus, of mammals that have been infected with an HCV. Preferredmammals include mice, chimpanzees, baboons, and humans. The HCV servesto expand the number of activated HCV-specific T cells in the mammal.The HCV-specific T cells derived from the mammal can then berestimulated in vitro by adding HCV NS3NS4NS5a or NS3NS4NS5aNS5bepitopic peptides to the T cells. The HCV-specific T cells can then betested for, inter alia, proliferation, the production of IFN-γ, and theability to lyse target cells displaying NS3NS4NS5a or NS3NS4NS5aNS5bepitopes in vitro.

In a lymphoproliferation assay (see Example 4), HCV-activated CD4⁺ Tcells proliferate when cultured with an NS3, NS4, NS5a, NS5b,NS3NS4NS5a, or NS3NS4NS5aNS5b epitopic peptide, but not in the absenceof an epitopic peptide. Thus, particular NS3, NS4, NS5a, NS5b,NS3NS4NS5a and NS3NS4NS5aNS5b epitopes that are recognized byHCV-specific CD4⁺ T cells can be identified using a lymphoproliferationassay.

Similarly, detection of IFN-γ in HCV-specific CD8⁺ T cells after invitro stimulation with the above-described fusion proteins, orindividual components of these proteins, can be used to identify NS3,NS4, NS5a, NS5b, NS3NS4NS5a, and NS3NS4NS5aNS5b epitopes thatparticularly effective at stimulating CD8⁺ T cells to produce IFN-γ (seeExample 3).

Further, ⁵¹Cr release assays are useful for determining the level of CTLresponse to HCV. See Cooper et al. Immunity 10:439-449. For example,HCV-specific CD8⁺ T cells can be derived from the liver of an HCVinfected mammal. These T cells can be tested in ⁵¹Cr release assaysagainst target cells displaying, e.g., NS3NS4NS5a NS3NS4NS5aNS5bepitopes. Several target cell populations expressing differentNS3NS4NS5a or NS3NS4NS5aNS5b epitopes can be constructed so that eachtarget cell population displays different epitopes of NS3NS4NS5a orNS3NS4NS5aNS5b. The HCV-specific CD8⁺ cells can be assayed against eachof these target cell populations. The results of the ⁵¹Cr release assayscan be used to determine which epitopes of NS3NS4NS5a or NS3NS4NS5aNS5bare responsible for the strongest CTL response to HCV. NS3NS4NS5a fusionproteins or NS3NS4NS5aNS5b fusion proteins which contain the epitopesresponsible for the strongest CTL response can then be constructed usingthe information derived from the ⁵¹Cr release assays.

An NS3NS4NS5a or NS3NS4NS5aNS5b fusion protein or polynucleotideencoding such a fusion protein, as well as the individual components ofthese fusion proteins or polynucleotides, can be administered to amammal, such as a mouse, baboon, chimpanzee, or human, to activateHCV-specific T cells in vivo. Administration can be by any means knownin the art, including parenteral, intranasal, intramuscular orsubcutaneous injection, including injection using a biological ballisticgun (“gene gun”), as discussed above.

Preferably, injection of an NS3NS4NS5a or NS3NS4NS5aNS5b polynucleotide,or a compositions containing a combination of the individual componentsof the fusion polynucleotides, is used to activate T cells. In additionto the practical advantages of simplicity of construction andmodification, injection of NS3NS4NS5a or NS3NS4NS5aNS5b polynucleotidesresults in the synthesis of an NS3NS4NS5a fusion protein orNS3NS4NS5aNS5b, respectively, in the host. Similarly, administration ofthe individual components of these polynucleotides, such as in acomposition consisting essentially of individual polynucleotidesencoding NS3, NS4, NS5a or a composition consisting essentially ofindividual polynucleotides encoding NS3, NS4, NS5a and NS5b, results inthe expression of the individual proteins in the host. Thus, theseimmunogens are presented to the host immune system with nativepost-translational modifications, structure, and conformation. Thepolynucleotides are preferably injected intramuscularly to a largemammal, such as a human, at a dose of 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5or 10 mg/kg.

A composition of the invention comprising an NS3NS4NS5a fusion proteinor polynucleotide, an NS3NS4NS5aNS5b fusion protein or polynucleotide,combinations of these fusions, or a combination of the individualcomponents thereof, is administered in a manner compatible with theparticular composition used and in an amount which is effective toactivate HCV-specific T cells as measured by, inter alia, a ⁵¹Cr releaseassay, a lymphoproliferation assay, or by intracellular staining forIFN-γ. The proteins and/or polynucleotides can be administered either toa mammal which is not infected with an HCV or can be administered to anHCV-infected mammal. The particular dosages of the polynucleotides orfusion proteins in a composition or will depend on many factorsincluding, but not limited to the species, age, and general condition ofthe mammal to which the composition is administered, and the mode ofadministration of the composition. An effective amount of thecomposition of the invention can be readily determined using onlyroutine experimentation. In vitro and in vivo models described above canbe employed to identify appropriate doses. The amount of NS3NS4NS5apolynucleotide used in the example described below provides generalguidance which can be used to optimize the activation of HCV-specific Tcells either in vivo or in vitro. Generally, 0.5, 0.75, 1.0, 1.5, 2.0,2.5, 5 or 10 mg of an NS3NS4NS5a or NS3NS4NS5aNS5b fusion protein orpolynucleotide, or of each of the individual components, will beadministered to a large mammal, such as a baboon, chimpanzee, or human.If desired, co-stimulatory molecules or adjuvants can also be providedbefore, after, or together with the compositions.

Immune responses of the mammal generated by the delivery of acomposition of the invention, including activation of HCV-specific Tcells, can be enhanced by varying the dosage, route of administration,or boosting regimens. Compositions of the invention may be given in asingle dose schedule, or preferably in a multiple dose schedule in whicha primary course of vaccination includes 1-10 separate doses, followedby other doses given at subsequent time intervals required to maintainand/or reenforce an immune response, for example, at 1-4 months for asecond dose, and if needed, a subsequent dose or doses after severalmonths.

III. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Those of skill in the art will readily appreciate that the invention maybe practiced in a variety of ways given the teaching of this disclosure.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXAMPLE 1 Production of NS3NS4NS5a Polynucleotides

A polynucleotide encoding NS3NS4NS5a (approximately amino acids 1027 to2399, numbered relative to HCV-1) (also termed “NS345a” herein) or NS5a(approximately amino acids 1973 to 2399, numbered relative to HCV-1) wasisolated from an HCV. Polynucleotides encoding a methionine residue wereligated to the 5′ end of these polynucleotides and the polynucleotideswere cloned into plasmid, vaccinia virus, and adenovirus vectors.

Immunization Protocols. In one immunization protocol, mice wereimmunized with 50 μg of plasmid DNA encoding either NS5a or encoding anNS3NS4NS5a fusion protein by intramuscular injection into the tibialisanterior. A booster injection of 10⁷ pfu of vaccinia virus (VV)-NS5a(intraperitoneal) or 50 μg of plasmid control (intramuscular) wasprovided 6 weeks later.

In another immunization protocol, mice were injected intramuscularly inthe tibialis anterior with 10¹⁰ adenovirus particles encoding anNS3NS4NS5a fusion protein. An intraperitoneal booster injection of 10⁷pfu of VV-NS5a or an intramuscular booster injection of 10¹⁰ adenovirusparticles encoding NS3NS4NS5a was provided 6 weeks later.

EXAMPLE 2

Immunization with DNA encoding an NS3NS4NS5a fusion protein activatesHCV-specific CD8⁺ T cells.

⁵¹Cr Release Assay. A ⁵¹Cr release assay was used to measure the abilityof HCV-specific T cells to lyse target cells displaying an NS5a epitope.Spleen cells were pooled from the immunized animals. These cells wererestimulated in vitro for 6 days with the CTL epitopic peptide p214K9(2152-HEYPVGSQL-2160; SEQ ID NO:1) from HCV-NS5a in the presence ofIL-2. The spleen cells were then assayed for cytotoxic activity in astandard ⁵¹Cr release assay against peptide-sensitized target cells(L929) expressing class I, but not class II MHC molecules, as describedin Weiss (1980) J. Biol. Chem. 255:9912-9917. Ratios of effector (Tcells) to target (B cells) of 60:1, 20:1, and 7:1 were tested. Percentspecific lysis was calculated for each effector to target ratio.

The results of the assays are shown in Tables 1 and 2. Table 1demonstrates that immunization with plasmid DNA encoding an NS3NS4NS5afusion protein activates CD8⁺ T cells which recognize and lyse targetcells displaying an NS5a epitope. Surprisingly the NS5a polypeptide ofthe NS3NS4NS5a fusion protein was able to activate T cells even thoughthe NS5a polypeptide was present in a fusion protein.

Similarly, Table 2 demonstrates that delivery of the NS3NS4NS5a fusionprotein to mice by means of an adenovirus vector also activates CD8⁺ Tcells which recognize and lyse target cells displaying an HCV NS5aepitope. Thus, immunization with either “naked” (plasmid) DNA encodingan NS3NS4NS5a fusion protein or adenovirus vector-encoded fusion proteincan be used to activate HCV-specific T cells.

EXAMPLE 3

Immunization with DNA encoding an NS3NS4NS5a fusion protein activatesHCV-specific CD8⁺ T cells which express IFN-γ.

Intracellular Staining for Interferon-gamma (IFN-γ). Intracellularstaining for IFN-γ was used to identify the CD8⁺ T cells that secreteIFN-γ after in vitro stimulation with the NS5a epitope p214K9. Spleencells of individual immunized mice were restimulated in vitro eitherwith p214K9 or with a non-specific peptide for 6-12 hours in thepresence of IL-2 and monensin. The cells were then stained for surfaceCD8⁺ and for intracellular IFN-γ and analyzed by flow cytometry. Thepercent of CD8⁺ T cells which were also positive for IFN-γ was thencalculated. The results of these assays are shown in Tables 1 and 2.Table 1 demonstrates that CD8⁺ T cells activated in response toimmunization with plasmid DNA encoding an NS3NS4NS5a fusion protein alsoexpress IFN-γ. Immunization with an NS3NS4NS5a fusion protein encoded inan adenovirus also results in CD8⁺ HCV-specific T cells which expressIFN-γ, although to a lesser extent than immunization with aplasmid-encoded NS3NS4NS5a fusion protein (Table 2).

TABLE 1 HCV-NS5a-Specific CD8+ T Cells in Mice Immunized with NS5a orNS345a DNA ⁵¹Cr Release Assay Intracellular Staining for IFN-γ PercentSpecific Lysis of Targets* Percent of CD8+ T Cells Positive for IFN-g**NS5a DNA NS345a DNA NS5a DNA NS345a DNA E:T ratio p214K9 — p214K9 —p214K9 — p214K9 — 60:1 77 5 66 6 20:1 61 4 49 2 1.74 0.26 1.18 0.40  7:129 1 29 4 *Target cells (L929) were pulsed with p214K9 or media aloneand labeled with ⁵¹Cr. **Spleen cells were cultured with p214K9 or mediaalone for 12 hours in the presence of monensin. p214K9 is a CTL epitopicpeptide (2152-HEYPVGSQL-2160, SEQ ID NO:1) from HCV-NS5a ‘—’ refers tothe absence of peptide

TABLE 2 HCV-NS5a-Specific CD8+ T Cells Primed by Adenovirus or DNAEncoding for NS345a ⁵¹Cr Release Assay Intracellular Staining for IFN-γPercent Specific Lysis of Targets* Percent of CD8+ T Cells Positive forIFN-g** NS5a DNA NS345a DNA NS5a DNA NS345a DNA E:T ratio p214K9 —p214K9 — p214K9 p214J p214K9 p214J 60:1 76 2 55 5 20:1 85 2 22 3 3.240.13 0.25 0.09  7:1 62 <1 10 3 *Target cells (L929) were pulsed withp214K9 or p214J and labeled with ⁵¹Cr. **Spleen cells were cultured withp214K9 or p214J for 12 hours in the presence of monensin. p214K9 is aCTL epitopic peptide (2152-HEYPVGSQL-2160, SEQ ID NO:1) from HCV-NS5ap214J is a control peptide (10 mer) from HCV-NS5a

EXAMPLE 4

Immunization with DNA encoding an NS3NS4NS5a fusion protein stimulatesproliferation of HCV-specific CD4⁺ T cells.

Lymphoproliferation assay. Spleen cells from pooled immunized mice weredepleted of CD8⁺ T cells using magnetic beads and were cultured intriplicate with either p222D, an NS5a-epitopic peptide from HCV-NS5a(2224-AELIEANLLWRQEMG-2238; SEQ ID NO:2), or in medium alone. After 72hours, cells were pulsed with 1 μCi per well of ³H-thymidine andharvested 6-8 hours later. Incorporation of radioactivity was measuredafter harvesting. The mean cpm was calculated.

As shown in Table 3, immunization with a plasmid-encoded NS3NS4NS5afusion protein stimulates proliferation of CD4⁺ HCV-specific T cells.Immunization with an adenovirus vector encoding the fusion protein alsoresulted in stimulated proliferation of CD4⁺ HCV-specific T cells (Table4).

TABLE 3 HCV-NS5a-Specific CD4+ T Cells in Mice Immunized with NS5a orNS345a DNA Mean CPM NS5a DNA NS345a DNA p222D media p222D media 4523 7404562 861 (x6.1) (x5.3) p222D is a CD4+ epitopic peptide (aa:2224-AELIEANLLWRQEMG-2238, SEQ ID NO: 2) from HCV-NS5a

TABLE 4 HCV-NS5-Specific CD4+ T Cells Primed by Adenovirus or DNAEncoding for NS345a Mean CPM NS345a Adeno NS345a DNA p222D media p222Dmedia 896 357 1510 385 (x2.5) (x3.9) p222D is a CD4+ epitopic peptide(aa: 2224-AELIEANLLWRQEMG-2238, SEQ ID NO: 2) from HCV-NS5a

EXAMPLE 5

Efficiency of NS345a-encoding DNA Vaccine Formulations to prime CTLs inmice.

Mice were immunized with either 10-100 μg of plasmid DNA encoding NS345afusion protein as described in Example 1, with PLG-linked DNA encodingNS345a, described below, or with DNA encoding NS345a, delivered viaelectroporation (see, e.g., International Publication No. WO/0045823 forthis delivery technique). The immunizations were followed by a boosterinjection 6 weeks later of 1×10⁷ pfu vaccinia virus encoding NS5a,plasmid DNA encoding NS345a or plasmid DNA encoding NS5a each asdescribed in Example 1.

PLG-delivered DNA. The polylactide-co-glycolide (PLG) polymers wereobtained from Boehringer Ingelheim, U.S.A. The PLG polymer used in thisstudy was RG505, which has a copolymer ratio of 50/50 and a molecularweight of 65 kDa (manufacturers data). Cationic microparticles withadsorbed DNA were prepared using a modified solvent evaporation process,essentially as described in Singh et al., Proc. Natl. Acad. Sci. USA(2000) 97:811-816. Briefly, the microparticles were prepared byemulsifying 10 ml of a 5% w/v polymer solution in methylene chloridewith 1 ml of PBS at high speed using an IKA homogenizer. The primaryemulsion was then added to 50ml of distilled water containing cetyltrimethyl ammonium bromide (CTAB) (0.5% w/v). This resulted in theformation of a w/o/w emulsion which was stirred at 6000 rpm for 12 hoursat room temperature, allowing the methylene chloride to evaporate. Theresulting microparticles were washed twice in distilled water bycentrifugation at 10,000 g and freeze dried. Following preparation,washing and collection, DNA was adsorbed onto the microparticles byincubating 100 mg of cationic microparticles in a 1 mg/ml solution ofDNA at 4 C. for 6 hours. The microparticles were then separated bycentrifugation, the pellet washed with TE buffer and the microparticleswere freeze dried.

CTL activity and IFN-γ expression were measured by ⁵¹Cr release assay orintracellular staining as described in examples 2 and 3 respectively.The results are shown in Table 5.

Results demonstrate that immunization using plasmid DNA encoding forNS345a to prime mice results in activation of CD8⁺ HCV specific T cells.

TABLE 5 Efficiency of NS345a-Encoding DNA Vaccine Formulations to PrimeCTLs in Mice ICS for IFN-gamma (% CD8+ cells that are IFN-g+) foldincrease NS345a DNA # of mice % # of vs. ‘naked’ CTL Vaccines Boost MeanSdtdevP tested responding expts DNA activity? NS345a DNA VVNS5a 1.021.70 41 68% 10 N/A YES NS345a DNA NS345a DNA 0.02 0.04 22  5% 5 N/A YESNS345a DNA NS5a DNA 0.22 0.21 24 63% 5 N/A YES NS345a DNA VVNS5a 5.004.36 7 100%  2 4.90 YES eV (electro- poration) PLGNS345a VVNS5a 2.652.54 6 100%  2 2.60 YES DNA PLGNS345a NS5aDNA 0.33 0.24 15 80% 3 1.50YES DNA

EXAMPLE 6 Immunization Routes and Replicon Particles SINCR (DC+)Encoding for NS345a

Alphavirus replicon particles, for example, SINCR (DC+) were prepared asdescribed in Polo et al., Proc. Natl. Acad. Sci. USA (1999)96:4598-4603. Mice were injected with 5×10⁶ IU SINCR (DC+) repliconparticles encoding for NS345a intramuscularly (IM) as described inExample 1, or subcutaneously (S/C) at the base of the tail (BoT) andfoot pad (FP), or with a combination of ⅔ of the DNA delivered via IMadministration and 1/3 via a BoT route. The immunizations were followedby a booster injection of vaccinia virus encoding NS5a as described inExample 1.

IFN-γ expression was measured by intracellular staining as described inExample 3. The results are shown in Table 6. The results demonstratethat immunization via SINCR (DC+) replicon particles encoding for NS345aby a variety of routes results in CD8⁺ HCV specific T cells whichexpress IFN-γ.

TABLE 6 Immunization Routes and SINCR (DC+) Replicon Particles EncodingNS345a (all mice VVNS5a challenged) ICS for IFN-gamma (% CD8+ cells thatare IFN-g+) # of mice % responding Vaccines Immunization Route MeanSdtdevP tested # of expts mice SINCR (DC+) 5 × 10⁶ 100% IM (ta) 1.110.63 3 1 100% SINCR (DC+) 5 × 10⁶ 100% S/C (BoT + FP) 0.62 0.29 3 1 100%SINCR (DC+) 5 × 10⁶ ⅔ IM (ta) + ⅓ S/C (BoT) 2.43 2.00 3 1 100%

EXAMPLE 7 SINCR (DC+) Vs SINDC (LP) Replicon Particles Encoding forNS345a

Alphavirus replicon particles, for example, SINCR (DC+) and SINCR (LP)were prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA(1999) 96:4598-4603. Mice were immunized with 1×10³ to 1×10⁷ IU of SINCR(DC+) or SINCR (LP) replicon particles encoding for NS345a, byintramuscular injection into the tibialis anterior, followed by abooster injection of 10⁷ pfu vaccinia virus encoding NS5a at 6 weeks.

IFN-γ expression was measured by intracellular staining as described inExample 3. Administration of an increase in the number of SINCR (DC+)replicon particles encoding NS345a resulted in an increase in % of CD8⁺T cells expressing IFN-γ.

EXAMPLE 8 Alphavirus Replicon Priming, Followed by Various BoostingRegimes

Alphavirus replicon particles, for example, SINCR (DC+) were prepared asdescribed in Polo et al., Proc. Natl. Acad. Sci. USA (1999)96:4598-4603. Mice were primed with SINCR (DC+), 1.5×10⁶ IU repliconparticles encoding NS345a, by intramuscular injection into the tibialisanterior, followed by a booster of either 10-100 μg of plasmid DNAencoding for NS5a, 10¹⁰ adenovirus particles encoding NS345a, 1.5×10⁶IUSINCR (DC+) replicon particles encoding NS345a, or 107 pfu vacciniavirus encoding NS5a at 6 weeks.

IFN-γ expression was measured by intracellular staining as described inExample 3. The results are shown in Table 7. The results demonstratethat boosting with vaccinia virus encoding NS5a DNA results in thestrongest generation of CD8⁺ HCV specific T cells which express IFN-γ.Boosting with plasmid encoding NS5a DNA also results in a good response,while lesser responses are noted with adenovirus NS345a or SINCRDC+boosted animals.

TABLE 7 Alphavirus Replicon Particle Priming, Followed by VariousBoosting Regimens ICS for IFN-gamma (% CD8+ cells that are IFN-g+) # ofmice % responding Vaccines Boost Mean SdtdevP tested # of expts miceSINCR (DC+) NS5a DNA 0.46 0.36 4 1 75% 1.5 × 10⁶ SINCR (DC+) AdenoNS345a (10 × 10¹⁰) 0.04 0.04 4 1 25% 1.5 × 10⁶ SINCR (DC+) SINCR (DC+)1.5 × 10⁶ 0.06 0.06 8 2 25% 1.5 × 10⁶ SINCR (DC+) VVNS5a (1 × 10⁷) 2.432.45 4 1 100%  1.5 × 10⁶

EXAMPLE 9 Alphaviruses Expressing NS345a

Alphavirus replicon particles, for example, SINCR (DC+) and SINCR (LP)were prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA(1999) 96:4598-4603. Mice were immunized with 1×10² to 1×10⁶ IU SINCR(DC+) replicons encoding NS345a via a combination of delivery routes (⅔IM and ⅓ S/C) as well as by S/C alone, or with 1×10² to 1×10⁶ IU SINCR(LP) replicon particles encoding NS345a via a combination of deliveryroutes (⅔ IM and ⅓ S/C) as well as by S/C alone. The immunizations werefollowed by a booster injection of 10⁷ pfu vaccinia virus encoding NS5aat 6 weeks.

IFN-γ expression was measured by intracellular staining as described inExample 3. The results are shown in FIG. 1. The results indicateactivation of CD8⁺ HCV specific T cells.

EXAMPLE 10 Efficiency of NS5a Encoding DNA Vaccine Formulations to PrimeCTLs in Mice

Mice were immunized with either 10-100 μg of plasmid DNA encoding NS5aas described in Example 1 or with PLG-linked DNA encoding NS5a asdescribed in Example 5. The immunizations were followed by a boosterinjection at 6 weeks of either 10-100 μg of plasmid DNA encoding forNS5a, 10¹⁰ adenovirus particles encoding NS345a, 1.5×10⁶ IU SINCR (DC+)replicon particles encoding NS345a, or 10⁷ pfu vaccinia virus encodingNS5a.

CTL activity and IFN-γ expression were measured by the methods describedin Examples 2 and 3.

The results are shown in Table 8. The results demonstrate that primingwith plasmid DNA encoding for NS5a or PLG-linked DNA encoding NS5aresults in activation of CD8⁺ HCV specific T cells.

TABLE 8 Efficiency of NS5a-Encoding DNA Vaccine Formulations to PrimeCTLs in Mice ICS for IFN-gamma (% CD8+ cells that are IFN-g+) foldincrease # of mice % # of vs. ‘naked’ CTL NS5a Vaccines Boost MeanSdtdevP tested responding expts DNA activity? NS5a DNA VVNS5a 1.67 1.498 100%  3 N/A YES NS5a DNA NS5a DNA 0.17 0.09 12 83% 3 N/A YES PLGNS5aNS5a DNA 0.22 0.09 9 100%  2 1.29 YES DNA NS5a DNA AdenoNS345a 0.10 0.084 50% 1 N/A NO NS5a DNA SINCRNS345a 0.20 0.17 4 75% 1 N/A YES

EXAMPLE 11 Efficiency of NS345b-encoding DNA Vaccine Formulations toPrime CTLs in Mice

Mice were immunized with 10-100 μg of plasmid DNA encoding NS34b byintramuscular injection to the tibialis anterior or with PLG linked DNAencoding NS5a as described in Example 5. The immunizations were followedby a booster injection of plasmid DNA encoding for NS5a as described inExample 1.

CTL activity and IFN-γ expression were measured by the methods describedin Examples 2 and 3.

The results are shown in Table 9. The results demonstrate that primingwith plasmid DNA encoding NS345b or PLG-linked NS345b results inactivation of CD8⁺ HCV specific T cells.

TABLE 9 Efficiency of NS345a-Encoding DNA Vaccine Formulations to PrimeCTLs in Mice ICS for IFN-gamma (% CD8+ cells that are IFN-g+) foldincrease NS345 DNA # of mice % # of vs. ‘naked’ CTL Vaccines Boost MeanSdtdevP tested responding expts DNA activity? NS345a DNA NS5a DNA 0.180.16 15 60%  3 N/A YES PLGNS345 DNA NS5a DNA 0.30 0.33 14 57% 3 1.67 YES

EXAMPLE 12 Administration of DNA via Separate Plasmids

Mice were immunized with 100 ug plasmid DNA encoding for NS345a or with100 μg PLG-linked DNA encoding NS345a. Additionally, separate DNAplasmids encoding NS5a, NS34a, and NS4ab (33.3 μg each) wereadministered concurrently to another group of mice. Finally, PLG-linkedDNA encoding NS5a, NS34a, and NS4ab (33.3 μg each) were administeredconcurrently to another group of mice. The immunizations were followedby a booster injection of 1×10⁷ pfu vaccinia virus encoding NS5a, 6weeks post first immunization.

IFN-γ expression was measured by the method described in Example 3. Theresults are shown in FIG. 2. The results demonstrate a particularlyvigorous response in the activation of CD8+ HCV specific T cells whenthe DNA is broken down into smaller sub units and linked to PLG.

Thus, HCV polypeptides; either alone or as fusions, to stimulatecell-mediated immune responses, are disclosed. Although preferredembodiments of the subject invention have been described in some detail,it is understood that obvious variations can be made without departingfrom the spirit and the scope of the invention as defined by theappended claims.

2 1 9 PRT Artificial Sequence Description of Artificial Sequence CTLepitopic peptide from HCV NS5a 1 His Glu Tyr Pro Val Gly Ser Gln Leu 1 52 15 PRT Artificial Sequence Description of Artificial Sequence CD4+epitopic peptide from HCV NS5a 2 Ala Glu Leu Ile Glu Ala Asn Leu Leu TrpArg Gln Glu Met Gly 1 5 10 15

What is claimed is:
 1. A fusion protein comprising hepatitis C virus (HCV) proteins, wherein said HCV proteins consist of an NS3, an NS4, and an NS5a polypeptide of an HCV with one or more epitopes selected from the group consisting of an epitope shown in SEQ ID NO:1 and an epitope shown in SEQ ID NO:2.
 2. A fusion protein comprising hepatitis C virus (HCV) proteins, wherein said HCV proteins consist of an NS3, an NS4, an NS5a, and NS5b polypeptide of an HCV with one or more epitopes selected from the group consisting of an epitope shown in SEQ ID NO:1 and an epitope shown in SEQ ID NO:2.
 3. A fusion protein according to either of claims 1 or 2, wherein one of the HCV polypeptides is from a different strain of HCV than the other HCV polypeptides.
 4. The fusion protein of claim 3 wherein each of the HCV polypeptides is from a different strain of HCV.
 5. A composition comprising: (a) a fusion protein according to either of claims 1 or 2; and (b) a pharmaceutically acceptable excipient.
 6. A composition comprising: (a) a fusion protein according to claim 4; and (b) a pharmaceutically acceptable excipient.
 7. A method of activating T cells which recognize an epitope of an HCV polypeptide, comprising the step of: contacting T cells with a fusion protein of either of claims 1 or 2, whereby a population of T cells are activated that recognizes an epitope selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2.
 8. The method of claim 7 wherein the T cells are obtained from a mammal selected from the group consisting of a mouse, a baboon, a chimpanzee, and a human.
 9. The method of claim 8 wherein the mammal is infected with an HCV.
 10. The method of claim 8 wherein the mammal is not infected with an HCV.
 11. The method of claim 7 wherein the population of T cells comprises CD4⁺ T cells.
 12. The method of claim 7 wherein the population of T cells comprises CD8⁺ T cells.
 13. The method of claim 12 wherein the CD8⁺ T cells express interferon-γ.
 14. The method of claim 12 wherein the CD8⁺ T cells specifically recognize an epitope of an NS5a polypeptide.
 15. The method of claim 14 wherein the epitope is selected from the group consisting of the epitopes shown in SEQ ID NO:1 and SEQ ID NO:2.
 16. The method of claim 7 wherein the T cells comprise CD8⁺ and CD4⁺ T cells.
 17. The method of claim 7 wherein the step of contacting further comprises contacting the T cells with an adjuvant.
 18. The fusion protein of claim 1, wherein the one or more epitopes is an epitope shown in SEQ ID NO:1.
 19. The fusion protein of claim 1, wherein the one or more epitopes is the epitope shown in SEQ ID NO:2.
 20. The fusion protein of claim 1, wherein the one or more epitopes are epitopes shown in SEQ ID NO:1 and SEQ ID NO:2.
 21. The fusion protein of claim 2, wherein the one or more epitopes is an epitope shown in SEQ ID NO:1.
 22. The fusion protein of claim 2, wherein the one or more epitopes is the epitope shown in SEQ ID NO:2.
 23. The fusion protein of claim 2, wherein the one or more epitopes are epitopes shown in SEQ ID NO:1 and SEQ ID NO:2.
 24. The method of claim 23 wherein the population of T cells recognizes an epitope shown in SEQ ID NO:1.
 25. The method of claim 7 wherein the population of T cells recognizes an epitope shown in SEQ ID NO:2.
 26. The method of claim 7 wherein the population of T cells recognizes an epitope shown in SEQ ID NO:1 and SEQ ID NO:2. 